Glioblastoma (GBM) is the most common and lethal brain tumor, with resistance to standard treatments such as surgery, radiation, and chemotherapy. This resistance stems in large part from two sources: 1) genetic heterogeneity that lets it survive inhibition of single signaling pathways and 2) a stem cell-like subpopulation f GBM stem cells (GSCs) that appear to generate the bulk of the cancer cells and are particularly stubborn targets. This Project attempts to address both problems through targeting novel signaling hubs in cancer, the diacylglycerol kinases (DGKs). Our prior studies of a microRNA cytotoxic to GBM cells led us to identify its knockdown of DGK? as a major driver of its cytotoxicity, indicating the potential utility of targeting this kinase. DGK? and its product phosphatidic acid had already been found important in numerous signaling pathways with oncogenic roles, further supporting the potential of DGKs as targets. We recently reported that knockdown and small-molecule inhibition of DGK? causes apoptotic cell death in GBM and GSC lines, as well as in other cancers, both in vitro and in mouse models. These studies also indicated antiangiogenic effects in vivo and the importance of mTOR and HIF-1? as mediators of DGK? effects in cancer. Since the prior submission of this application and our published report, we have discovered that an abandoned medication found safe in prior clinical trials for a non-cancer indication, ritanserin, is a novel DGK inhibitor. We hypothesize that the DGKs are promising therapeutic targets in GBM, and that repurposing ritanserin will allow rapid clinical translation of this strategy. This hypothesis will be investigated in depth with the proposed studies. In Aim 1, we will assess whether multiple DGKs have important overlapping functions in GSCs, and whether the DGK? role is unique among DGK family members. Aim 2 will test if the effects of ritanserin and an established DGK? inhibitor on GSCs are mediated largely by inhibition of mTOR and HIF-1?. The studies of Aim 3 will determine pharmacokinetics, safety, and efficacy of ritanserin in GSC xenografts and a transgenic mouse model of high-grade glioma, while Aim 4 will evaluate whether even greater efficacy can be achieved with delivery of DGK inhibitors by a sustained-release pump or by an innovative technique involving polymer-coated microbubbles/focused ultrasound. Successful completion of the proposed studies will shed light on the biology and therapeutic targeting of the DGKs in GBM, with the potential for rapid translation to clinical trials. This strategy may have broad applicability in cancer, acting ia direct cytotoxicity to cancer cells, antiangiogenic effects, and enhancement of other therapies; recent reports indicate that DGK inhibition is a promising approach to enhancing cancer immunotherapy.